Networks of branched actin filaments are highly dynamic structures and their assembly is hypothesized to generate the force for many types of cell motility including cell migration, intracellular transport, endocytosis and the movement of intracellular pathogens. The proteins involved in regulating the assembly of these networks have been conserved throughout eukaryotes. Using the yeast actin patch as a model system, this proposal aims to test predictions of the dendritic nucleation model of actin network assembly, which describes how networks of branched actin filaments are assembled and can generate force, in vivo. A central player in this model is the Arp2/3 complex, which generates branches and seeds the formation of new filaments. These studies will focus on understanding the molecular mechanisms by which regulators of Arp2/3 activity function to drive the assembly and movement of the yeast actin patch and endocytosis. The movement of GFP-labeled actin patch components, which undergo reproducible movements during specific phases of patch evolution, will be studied. The movement of patches will be examined in strains carrying mutations in Arp2/3 regulators and will be analyzed in large numbers using high-speed microscopy coupled with computer-aided particle tracking and quantitative motion analysis. In addition the architecture of the actin networks in these mutants will be analyzed by electron microscopy. The force required for cells to migrate, to move internal cargoes within, to inernalize materials from the outside and for the movement of some pathogenic bacteria is provided by the polymerization of networks of actin filaments. The proper regulation of the assembly of these networks is essential for normal cellular function and for normal development of mutlicellular organisms and the misregulation of these networks often accompanies diseases, like cancer. By understanding how the formation of actin networks is regulated in cells we will be able to better understand both the normal functions of cells and how their misregulation can lead to disease. [unreadable] [unreadable] [unreadable]